Infections caused by Staphylococcus aureus span a wide clinical spectrum, ranging from asymptomatic nasal carriage to endocarditis, bone and joint infections and lethal shock. Increasing rates of S. aureus infection and the emergence of community-acquired strains drive the need for an increased understanding of the virulence determinants of this emerging pathogen and evaluating the role they play in outcome of patients with S. aureus bacteremia. Evaluating the role of these virulence determinants in humans was limited by the absence of a large, well-characterized collection of bloodstream S. aureus isolates. Such a clinical resource was developed by Vance Fowler (Co-Investigator), when he created one of the world<s largest collections of prospectively characterized isolates from patients with S. aureus bacteremia (SAB). Stringent clinical grouping definitions were applied to this collection (the S. aureus bacteremia group or SABG), which minimized patient heterogeneity, a critical component in evaluating potential associations between clinical severity and bacterial virulence determinants. In the original R01 application, multilocus sequence typing (MLST) and array comparative genome hybridization (aCGH) were used to determine phylogenetic relationships and to identify genomic differences (e.g., presence or absence of virulence factors, mobile genetic elements, etc.) among a subset of 379 SABG isolates. Within this subset, two clonal complexes (CC5 and CC30) showed a significant trend toward more complicated infection. Array CGH analysis of SAGA indicates that isolates within CC5 and CC30 are associated with an increased frequency of mobile genetic elements (MGE) carrying virulence factors and that MGE contain a remarkable level of internal sequence divergence between isolates in different CC. Furthermore, nucleotide polymorphisms are found throughout the S. aureus genomes, but are enriched in MGE and bacterial surface proteins. Based on our results and other experimental data suggesting that genomic polymorphisms and rearrangements have significant roles in S. aureus virulence, we now propose to: 1) use the murine sepsis model to test hypotheses related to virulence of CC5 and CC30 and candidate virulence genes; 2) complete high-throughput genome sequencing and comparative analysis to identify genomic polymorphisms and novel genes in a subset of S. aureus isolates (complicated infection group: CIG); 3) complete genome wide expression analysis of CIG isolates in the murine sepsis model; 4) use genome wide association studies to identify genomic polymorphisms associated with virulence and differential gene expression and 4) complete allelic-replacement of selected virulence candidates and test in murine model. PUBLIC HEALTH RELEVANCE: Genetic factors in Staphylococcus aureus play a critical role in causing and determining the severity of infection and disease. This study proposes to identify these variations in a large collection of clinically isolated, disease-causing Staphylococci by examining genes known to be involved and identifying new genes that may be involved in infection. Knowledge acquired from this study could potentially be used to develop novel and possibly preventive therapies to eradicate complicated staphylococcal infections.